Nitric oxide-releasing medical devices

ABSTRACT

An implant or intravascular stent comprising a polymeric composition capable of releasing nitric oxide under physiological conditions. The polymeric composition comprises a polymer and at least one nitric oxide-releasing N 2 O 2   −  functional group bound to the polymer.

RELATED APPLICATION

This is a continuation of U.S. application Ser. No. 09/666,668, filedSep. 20, 2000, now U.S. Pat. No. 6,379,660, which is a continuation ofU.S. application Ser. No. 08/837,812, filed Apr. 22, 1997, now U.S. Pat.No. 6,200,558, which is a divisional of U.S. application Ser. No.08/344,157, filed on Nov. 22, 1994, now U.S. Pat. No. 5,632,981, whichis a continuation-in-part of U.S. application Ser. No. 08/121,169, filedon Sep. 14, 1993, now U.S. Pat. No. 5,525,357, which is acontinuation-in-part of U.S. application Ser. No. 07/935,565, filed onAug. 24, 1992, now U.S. Pat. No. 5,405,919. The entire disclosure of the'660, '558, '981, '357 and '919 patents are incorporated herein byreference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to polymeric compositions capable ofreleasing nitric oxide. In particular, the present invention relates topolymeric compositions comprising a biopolymer, such as a peptide,polypeptide, protein, oligonucleotide, nucleic acid, or the like towhich is bound a nitric oxide-releasing N₂O₂ ⁻ functional group,pharmaceutical compositions comprising such polymeric compositions, andmethods of treating biological disorders with such a biopolymericcomposition.

BACKGROUND OF THE INVENTION

Nitric oxide (NO) has recently been implicated in a variety ofbioregulatory processes, including normal physiological control of bloodpressure, macrophage-induced induced cytostasis and cytotoxicity, andneurotransmission (Moncada et al., “Nitric Oxide from L-Arginine: ABioregulatory System,” Excerpta Medica, International Congress Series897 (Elsevier Science Publishers B.V.: Amsterdam, 1990); Marletta etal., “Unraveling the Biological Significance of Nitric Oxide,”Biofactors, 2, 219-225 (1990); Ignarro, “Nitric Oxide. A Novel SignalTransduction Mechanism for Transcellular Communication,” Hypertension(Dallas), 16, 477-483 (1990)). A number of compounds have been developedwhich are capable of delivering nitric oxide, including compounds whichrelease nitric oxide upon being metabolized and compounds which releasenitric oxide spontaneously in aqueous solution.

Those compounds which release nitric oxide upon being metabolizedinclude the widely used nitrovasodilators glyceryl trinitrate and sodiumnitroprusside (Ignarro et al., J. Pharmacol. Exp. Ther., 218, 739-749(1981); Ignarro, Annu. Rev. Pharmacol. Toxicol., 30, 535-560 (1990);Kruszyna et al., Toxicol. Appl. Pharmacol., 91, 429-438 (1987); Wilcoxet al., Chem. Res. Toxicol., 3, 71-76 (1990). Another compound,S-nitroso-N-acetylpenicillamine, has been reported to release nitricoxide in solution and to be effective at inhibiting DNA synthesis (Garget al., Biochem. and Biophys. Res. Comm., 171, 474-479 (1990)).

Numerous nitric oxide-nucleophile complexes have been described, e.g.,Drago, ACS Adv. Chem. Ser., 36, 143-149 (1962). See also Longhi andDrago, Inog. Chem., 2, 85 (1963). Some of these complexes are known toevolve nitric oxide on heating or hydrolysis, e.g., Maragos et al., J.Med. Chem. 34, 3242-3247 (1991).

The cytostatic effect of nitric oxide solutions on tumor cells in vitrohas been demonstrated. In particular, it has been shown that solutionsof nitric oxide inhibit DNA synthesis and mitochondrial respiration oftumor cells in vitro (Hibbs et al., Biochem. and Biophys. Res. Comm.,157, 87-94 (1988); Stuehr et al., J. Exp. Med., 169, 1543-1555 (1989)).

Endothelium-derived relaxing factor (EDRF) is a labile humoral agentwhich is part of a cascade of interacting agents involved in therelaxation of vascular smooth muscle. EDRF is thus important in thecontrol of vascular resistance to blood flow and in the control of bloodpressure. Some vasodilators act by causing EDRF to be released fromendothelial cells. (See Furchgott, Ann. Rev. Pharmacol. Toxicol., 24,175-197 (1984).) In 1987, Palmer et al., presented evidence that EDRF isidentical to the simple molecule, nitric oxide, NO (Nature, 317, 524-526(1987)), though more recently, that conclusion has been challenged(Myers et al., Nature, 345, 161-163, 1990)).

Nitric oxide in its pure form, however, is a highly reactive gas havinglimited solubility in aqueous media (WHO Task Group on EnvironmentalHealth Criteria for Oxides of Nitrogen, Oxides of Nitrogen,Environmental Health Criteria 4 (World Health Organization: Geneva,1977)). Nitric oxide, therefore, is difficult to introduce reliably intomost biological systems without premature decomposition.

The difficulty in administering nitric oxide can be overcome in somecases by administering nitric oxide pharmacologically in prodrug form.The compounds glyceryl trinitrate and sodium nitroprusside arerelatively stable and release nitric oxide only on activation (Ignarroet al., J. Pharmacol. Exp. Ther., 218, 739-749 (1981); Ignarro, Annu.Rev. Pharmacol. Toxicol., 30, 535-560 (1990); Kruszyna et al., Toxicol.Appl. Pharmacol., 91, 429-438 (1987); Wilcox et al., Chem. Res.Toxicol., 3, 71-76 (1990)). While this feature may be an advantage insome applications, it can also be a significant liability, as in thedevelopment of tolerance to glyceryl trinitrate via the exhaustion ofthe relevant enzyme/cofactor system (Ignarro et al., Annu. Rev.Pharmacol. Toxicol., 25, 171-191 (1985); Kuhn et al., J. Cardiovasc.Pharmacol., 14 (Suppl. 11), S47-S54 (1989)) and toxicity frommetabolically produced cyanide during prolonged administration ofnitroprusside (Smith et al., “A Potpourri of Biologically ReactiveIntermediates” in Biological Reactive Intermediates IV. Molecular andCellular Effects and Their Impact on Human Health (Witmer et al., eds.),Advances in Experimental Medicine and Biology Volume 283 (Plenum Press:New York, 1991), pp. 365-369).

Evidence that nitric oxide is released from the endothelial cells and isresponsible for the relaxation of the vascular smooth muscle, and hencethe control of blood pressure, has resulted in the development ofartificial agents that can deliver nitric oxide in vivo. A veryimportant class of such agents is the nitric oxide-nucleophilecomplexes. Recently, a method for treating cardiovascular disorders in amammal with certain nitric oxide-nucleophile complexes was disclosed,e.g. in U.S. Pat. No. 4,954,526. These compounds contain the anionicN₂O₂ ⁻ group or derivatives thereof. See also, Maragos et al., J. Med.Chem., 34, 3242-3247 (1991). Many of these compounds have provenespecially promising pharmacologically because, unlike nitrovasodilatorssuch as nitroprusside and nitroglycerin, they release nitric oxidewithout first having to be activated. The only other series of drugscurrently known to be capable of releasing nitric oxide purelyspontaneously is the S-nitrosothiol series, compounds of structureR—S—NO (Stamler et al., Proc. Natl. Acad. Sci. U.S.A., 89, 444-448(1992); Stamler et al., Proc. Natl. Acad. Sci. U.S.A., 89, 8087-8091(1992)); however, the R—S—NO—NO reaction is kinetically complicated anddifficult to control (Morley et al., J. Cardiovasc. Pharmacol., 21,670-676 (1993)). The N₂O₂ ⁻ containing compounds are thus advantageousamong drugs currently known in that they decompose at any given pH via acleanly first order reaction to provide doses of nitric oxide that canbe predicted, quantified, and controlled. See, e.g., Maragos et al., J.Med. Chem., 34, 3242-3247 (1991).

Nitric oxide/nucleophile complexes which release nitric oxide in aqueoussolution are also disclosed in U.S. Pat. Nos. 5,039,705, 5,185,376,5,155,137, 5,208,233, 5,212,204, 5,250,550 and 5,366,997 (issue dateNov. 22, 1994) as well as in pending U.S. patent application Ser. Nos.07/764,908 (filed Sep. 24, 1991), Ser. No. 07/858,885 (filed Mar. 27,1992), Ser. No. 07/867,759 (filed Apr. 13, 1992), Ser. No. 07/935,565(filed Aug. 24, 1992), Ser. No. 08/017,270 (filed Feb. 12, 1993), andSer. No. 08/121,169 (filed Sep. 14, 1993) as useful therapeutic agents(see also Maragos et al., J. Med. Chem., 34, 3242-3247 (1991)).

Despite the promise of the nitric oxide/nucleophile adducts that havebeen investigated, their pharmacological application has been limited bytheir tendency to distribute evenly throughout the medium. Such evendistribution is a great advantage in many research applications, buttends to compromise their selectivity of action. Another limitation tothe application of these nitric oxide/nucleophile adducts is theirpropensity for relatively rapid release of nitric oxide which maynecessitate frequent dosing to achieve a prolonged biological effect.Thus there remains a need for nitric oxide-releasing compositions whichare capable of concentrating the effect of the nitric oxide release to asitus of application and for which nitric oxide release may becontrolled for effective dosing.

It is, therefore, a principal object of the present invention to providea polymeric composition comprising a biopolymer to which is bound a N₂O₂⁻ functional group and which is capable of releasing NO underphysiological conditions. Another object of the invention is to providea polymeric composition comprising a biopolymer to which is bound a N₂O₂⁻ functional group whose release of NO can be controlled such that localor cell/tissue specific release can be effected. It is another object ofthe present invention to provide a polymeric composition comprising abiopolymer to which is bound a N₂O₂ ⁻ functional group whose release ofNO is such that a prolonged biological effect can be attained. Yetanother object of the present invention is to provide pharmaceuticalcompositions comprising such biopolymeric compositions. It is also anobject of the present invention to provide a method of treating abiological disorder involving the administration of such biopolymericcompositions. These and other objects and advantages of the presentinvention, as well as additional inventive features, will be apparentfrom the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a polymeric composition capable ofspontaneously releasing nitric oxide under physiological conditions. Thepolymeric composition comprises a biopolymer to which is bound a nitricoxide-releasing N₂O₂ ⁻ functional group. “Biopolymer(ic)” is meant toinclude any biological polymer, such as peptides, polypeptides,proteins, oligonucleotides, and nucleic acids, including those thatcontain naturally occurring and/or nonnaturally occurring subunits.Specific examples include antibodies or fragments thereof and peptidehormones, proteins, and growth factors for which the target cell typehas a high population of receptors. The preferred nitric oxide-releasingN₂O₂ ⁻ functional group which is used to form the biopolymer-boundNONOates of the present invention is defined by the formula:

wherein X is an organic or inorganic moiety and X′ may be the same as X,or it may be a pharmaceutically acceptable metal center, apharmaceutically acceptable cation, or the like. The N₂O₂ ⁻ group isbonded to the biopolymer through either or both the linking groups X andX′.

By “bound to a polymer,” it is meant that the N₂O₂ ⁻ functional group isassociated with, part of, incorporated with or contained within thebiopolymer physically or chemically. Bonding of the N₂O₂ ⁻ functionalgroup to the polymer can be achieved by covalent bonding of the N₂O₂ ⁻group to the biopolymer through a linking group X or X′. Chemicalbonding of the N₂O₂ ⁻ functional group to the biopolymer may be by, forexample, covalent bonding of the linking group X or X′ to the biopolymersuch that the linking group forms part of the biopolymer itself, i.e.,is in the biopolymer backbone or is attached to pendant groups on thebiopolymer backbone. The manner in which the nitric oxide-releasing N₂O₂⁻ functional group is associated with, part of, incorporated with orcontained within, i.e., “bound,” to the polymer is inconsequential tothe present invention and all means of association, incorporation andbonding are contemplated herein.

In another aspect of the invention, the biopolymer-bound nitricoxide-releasing compositions of the present invention can be bound to orphysically associated with polymers that are not biopolymers (referredto hereinafter as “non-biopolymers”).

The present invention also provides a pharmaceutical composition whichincludes a pharmaceutically acceptable carrier and a polymericcomposition comprising a biopolymer to which is bound a nitricoxide-releasing N₂O₂ ⁻ functional group.

The invention further provides a method of treating biological disordersin which dosage with nitric oxide would be therapeutic which comprisesadministering to a mammal afflicted with such a biological disorder apolymeric composition, comprising a biopolymer to which is bound anitric oxide-releasing N₂O₂ ⁻ functional group, in an amount sufficientto release a therapeutically effective amount of nitric oxide.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is predicated on the discovery that usefulpharmacological agents can be provided by incorporating nitricoxide-releasing N₂O₂ ⁻ functional groups into a biopolymer. Accordingly,the N₂O₂ ⁻ functional group is “bound to the polymer” as that term hasbeen defined herein. The term NONOate is used herein as a shorthand torefer to the nitric oxide-releasing N₂O₂ ⁻ group.

It has been discovered that incorporation of a NONOate into a biopolymerprovides a biopolymer-bound NONOate composition that can be applied withspecificity to a biological site of interest. Site specific applicationof the biopolymer-bound NONOate enhances the selectivity of action ofthe nitric oxide-releasing NONOate. If N₂O₂ ⁻ functional groups attachedto the biopolymer are necessarily localized, then the effect of theirnitric oxide release will be concentrated in the tissues with which theyare in contact. If the biopolymer is soluble, selectivity of action canstill be arranged, for example, by attachment to or derivatization of anantibody specific to the target tissue. Similarly, attachment of N₂O₂ ⁻groups to small peptides that mimic the recognition sequences of ligandsfor important receptors provides localized concentrated effect of nitricoxide release, as would attachment to oligonucleotides capable ofsite-specific interactions with target sequences in a nucleic acid.Other proteins, peptides, polypeptides, nucleic acids andpolysaccharides, including hormones and motility, chemotactic andextravasating factors or agents, can be similarly utilized.

By way of illustration, a piperazine monoNONOate derivative can becovalently attached to a polypeptide containing the IKVAV recognitionsequence important in tumor cell chemotaxis. Through retention of boththe capacity to regenerate NO as an antichemotactic agent and theaffinity of the IKVAV sequence for tumor cells and/or sites in thevascular and lymphatic systems where the tumor cells tend to attach,metastasis can be reduced or even prevented.

While not being bound to any particular theory, it is believed thatlongevity of nitric oxide release in the biopolymer-bound NONOatecompositions of the present invention is to be attributed both to thephysical structure of the composition and to electrostatic effects.Thus, it is believed that if the biopolymer is an insoluble solid, N₂O₂⁻ groups near the surface of the particle should be available for rapidrelease while those that are more deeply imbedded are stericallyshielded, requiring more time and/or energy for the nitric oxide to workits way into the medium. Unexpectedly, it has been found that increasingpositive charge in the vicinity of an N₂O₂ ⁻ functional group also tendsto increase the halflife of nitric oxide generation. The mechanism ofthis rate retardation may be attributable simply to repulsiveelectrostatic interactions, i.e., increasing the number of H⁺-repellingpositive charges in the vicinity of the N₂O₂ ⁻ groups inhibits attack ofpositively charged H⁺ ions on the N₂O₂ ⁻ functional group and slows therate of its H⁺-catalyzed decomposition. For example, by attaching aminogroups to the polymeric support that are capable of forming the nitricoxide-releasing N₂O₂ ⁻ functional group on reaction with nitric oxide,partially converted structures can be produced on less-than-exhaustivetreatment with nitric oxide that after exposure to water contain a largenumber of positively charged ammonium centers surrounding the N₂O₂ ⁻group that electrostatically inhibit the approach of H⁺ ions capable ofinitiating nitric oxide loss from the nitric oxide-releasing N₂O₂ ⁻functional group.

The nitric oxide-releasing N₂O₂ ⁻ functional groups that are bound tothe biopolymer generally are capable of releasing nitric oxide in anaqueous environment spontaneously upon contacting an aqueousenvironment, i.e., they do not require activation through a redoxreaction or electron transfer such as is required for glyceryltrinitrate and sodium nitroprusside. Some of the nitricoxide/nucleophile complexes useful in the context of the presentinvention do require activation by particular means, but only asnecessary to free the nitric oxide-releasing X[N(O)NO]⁻ group in thevicinity of the particular cells of interest. As an example, covalentattachment of a protecting group to the anionic [N(O)NO]⁻ functionprovides a means of postponing nitric oxide release until the moleculereaches an organ capable of metabolically removing the protecting group.By choosing a protecting group that is selectively cleaved by enzymesspecific to a tumor, biological disorder, cell, or tissue of interest,for example, the action of the nitric oxide/nucleophile complex can betargeted to maximize the desired effect. While the biopolymer-boundNONOate compositions of the present invention are capable of releasingnitric oxide in an aqueous solution, such a compound preferably releasesnitric oxide under physiological conditions.

For example, a NONOate functionality can be attached to a tumor-specificantibody or other protein which has one or more lysine side chain aminogroups that are unnecessary to the function of the protein by reactingsaid lysine group(s) with a derivatizing agent capable of covalentlyattaching first to the lysine amino nitrogen then in a subsequent stepto the sulfur atom of an O-functionalized NONOate containing a freethiol grouping elsewhere in the molecule. Once such a protein arrives atthe desired target tissue after systemic application, enzymatic orhydrolytic removal of the substituent bound to oxygen frees the anionicNONOate function to concentrate NO release at that site.

The preferred nitric oxide-releasing N₂O₂ ⁻ functional group which isused to form the biopolymer-bound NONOates of the present invention isdefined by the formula:

wherein X is an organic or inorganic moiety and X′ is an organic orinorganic substituent, a pharmaceutically acceptable metal center, apharmaceutically acceptable cation, or the like. The N₂O₂ ⁻ group isbonded to the biopolymer through either or both the linking groups X andX′.

The nitric oxide-releasing N₂O₂ ⁻ functional group is preferably anitric oxide/nucleophile adduct, e.g., a complex of nitric oxide and anucleophile most preferably a nitric oxide/nucleophile complex whichcontains the anionic moiety X[N(O)NO]⁻, where X is any suitablenucleophile residue. The nucleophile residue is preferably that of aprimary amine (e.g., X=(CH₃)₂CHNH, as in (CH₃)₂CHNH[N(O)NO]Na), asecondary amine (e.g., X=(CH₃CH₂)₂N, as in (CH₃CH₂)₂N[N(O)NO]Na), apolyamine (e.g., X=spermine, as in the zwitterion H₂N(CH₂)₃NH₂⁺(CH₂)₄N[N(O)NO]⁻(CH₂)₃NH₂, X=2-(ethylamino)ethylamine, as in thezwitterion CH₃CH₂N[N(O)NO]⁻CH₂CH₂NH₃ ⁺, orX=3-(n-propylamino)propylamine, as in the zwitterionCH₃CH₂CH₂N[N(O)NO]⁻CH₂CH₂CH₂NH₃ ⁺), or oxide (i.e., X=O⁻, as inNaO[N(O)NO]Na), or a derivative thereof. Such nitric oxide/nucleophilecomplexes are capable of delivering nitric oxide in a biologicallyusable form as a predictable rate.

Other suitable nitric oxide/nucleophile complexes include those havingthe following formulas:

wherein J is an organic or inorganic moiety, M^(+x) is apharmaceutically acceptable cation, where x is the valence of thecation, a is an integer of at least one, and b and c are the smallestintegers that result in a neutral compound, as described in U.S. Pat.No. 5,208,233, and incorporated herein by reference;

wherein b and d are the same or different and may be zero or one, R₁,R₂, R₃, R₄, and R₅ are the same or different and may be hydrogen, C₃₋₈cycloalkyl, C₁₋₁₂ straight or branched chain alkyl, benzyl, benzoyl,phthaloyl, acetyl, trifluoroacetyl, p-toluyl, t-butoxycarbonyl, or2,2,2-trichloro-t-butoxycarbonyl, and x, y, and z are the same ordifferent and are integers from 2 to 12, as described in U.S. Pat. No.5,155,137, incorporated herein by reference;

and R₇ are the same or different and may be hydrogen, C₃₋₈ cycloalkyl,C₁₋₁₂ straight or branched chain alkyl, benzyl, benzoyl, phthaloyl,acetyl, trifluoroacetyl, p-toluyl, t-butoxycarbonyl, or2,2,2-trichloro-t-butoxycarbonyl, f is an integer from 0 to 12, with theproviso that when B is the substituted piperazine moiety

then f is an integer from 2 to 12, as described in U.S. Pat. No.5,155,137, incorporated herein by reference;

wherein R₈ is hydrogen, C₃₋₈ cycloalkyl, C₁₋₁₂ straight or branchedchain alkyl, benzyl, benzoyl, phthaloyl, acetyl, trifluoroacetyl,p-toluyl, t-butoxycarbonyl, or 2,2,2-trichloro-t-butoxycarbonyl, R₉ ishydrogen or a C₁-C₁₂ straight or branched chain alkyl, and g is 2 to 6,as described in U.S. Pat. No. 5,250,550, incorporated herein byreference;

wherein R₁ and R₂ are independently selected from the group consistingof a straight chain or branched chain C₁-C₁₂ alkyl group and a benzylgroup, or else R₁ and R₂, together with the nitrogen atom, are bonded toform a heterocyclic group, preferably a pyrrolidino, piperidino,piperazino or morpholino group, M^(+x) is a pharmaceutically acceptablecation, and x is the valence of the cation, as described in U.S. Pat.Nos. 5,039,705 and 5,208,233 and U.S. patent application Ser. No.08/017,270, filed Feb. 12, 1993, and incorporated here in by reference;K[(M)^(x′) _(x)(L)_(y)(R¹R²N—N₂O₂)_(z)]  (VI)wherein M is a pharmaceutically acceptable metal, or, where x is atleast two, a mixture of two different pharmaceutically acceptablemetals, L is a ligand different from (R¹R²N—N₂O₂) and is bound to atleast one metal, R¹ and R² are each organic moieties and may be the sameor different, x is an integer of from 1 to 10, x′ is the formaloxidation state of the metal M, and is an integer of from 1 to 6, y isan integer of from 1 to 18, and where y is at least 2, the ligands L maybe the same or different, z is an integer of from 1 to 20, and K is apharmaceutically acceptable counterion to render the compound neutral tothe extent necessary, as described in U.S. patent application Ser. No.07/858,885, filed Mar. 27, 1992, and incorporated herein by reference;[R—N(H)N(NO)O—]_(y)X  (VII)wherein R is C₂₋₈ lower alkyl, phenyl, benzyl, or C₃₋₈ cycoloalkyl, anyof which R groups may be substituted by one to three substituents, whichare the same or different, selected from the group consisting of halo,hydroxy, C₁₋₈ alkoxy, —NH₂, —C(O)NH₂, —CH(O), —C(O)OH, and —NO₂, X is apharmaceutically acceptable cation, a pharmaceutically acceptable metalcenter, or a pharmaceutically acceptable organic group selected from thegroup consisting of C₁₋₈ lower alkyl, —C(O)CH₃, and —C(O)NH₂, and y isone to three, consistent with the valence of X, as described in U.S.Pat. No. 4,954,526 and incorporated herein by reference; and

wherein R₁ and R₂ are independently chosen from C₁₋₁₂ straight chainalkyl, C₁₋₁₂ alkoxy or acyloxy substituted straight chain alkyl, C₂₋₁₂hydroxy or halo substituted straight chain alkyl, C₃₋₁₂ branched chainalkyl, C₃₋₁₂ hydroxy, halo, alkoxy, or acyloxy substituted branchedchain alkyl, C₃₋₁₂ straight chain olefinic and C₃₋₁₂ branched chainolefinic which are unsubstituted or substituted with hydroxy, alkoxy,acyloxy, halo or benzyl, or R₁ and R₂ together with the nitrogen atom towhich they are bonded form a heterocyclic group, preferably apyrrolidino, piperidino, piperazino or morpholino group, and R₃ is agroup selected from C₁₋₁₂ straight chain and C₃₋₁₂ branched chain alkylwhich are unsubstituted or substituted by hydroxy, halo, acyloxy oralkoxy, C₂₋₁₂ straight chain or C₃₋₁₂ branched chain olefinic which areunsubstituted or substituted by halo, alkoxy, acyloxcy or hydroxy, C₁₋₁₂unsubstituted or substituted acyl, sulfonyl and carboxamido; or R₃ is agroup of the formula —(CH₂)_(n)—ON═N(O)NR₁R₂, wherein n is an integer of2-8, and R₁ and R₂ are as defined above; with the proviso that R₁, R₂and R₃ do not contain a halo or a hydroxy substituent α to a heteroatom,as described in U.S. application Ser. No. 07/950,637, filed Sep. 23,1992.

Any of a wide variety of biopolymers can be used in the context of thepresent invention. Biopolymers suitable for use include peptides,polypeptides, proteins, oligonucleotides, nucleic acids, e.g., RNA andDNA, antibodies, peptide hormones, glycoproteins, glycogen, and thelike. Alternatively, a subunit of a biopolymer, such as a fatty acid,glucose, an amino acid, a succinate, a ribonucleotide, a ribonucleoside,a deoxyribonucleotide, and a deoxyribonucleoside can be used.Illustrative examples include antibodies or fragments thereof;extracellular matrix proteins such as laminin, fibronectin, or theircell attachment-site peptide recognition sequences, such as RGDS, IKVAV,YIGSR, and the like; and growth factors, peptide hormones, and otherpolypeptides for which there are high-affinity cell surface receptorsites, such as EGF, TGFα, TGFβ and TNF. Such molecules, upon receptorbinding, may be internalized into the target cells, thereby facilitatingintracellular delivery of the NO donor moiety.

The nitric oxide-releasing N₂O₂ ⁻ functional groups may be bound to thebiopolymer by formation of a nitric oxide/nucleophile complex of thetype and having the formulas of those described above, in situ on thebiopolymer. The N₂O₂ ⁻ functional group may be attached to an atom inthe backbone of the biopolymer, or it may be attached to a group pendantto the biopolymer backbone, or it may simply be entrapped in thebiopolymer matrix. Where the N₂O₂ ⁻ functional group is attached to thebiopolymer backbone, the biopolymer includes in its backbone sites whichare capable of reacting with nitric oxide to bind the nitric oxide forfuture release. For example, the biopolymer can include nucleophilicnitrogen atoms which react with nitric oxide to form the N₂O₂ ⁻functional group at the nitrogen in the backbone. Where the N₂O₂ ⁻functional group is a group pendant to the polymer backbone, thebiopolymer contains, or is derivatized with, a suitable pendantnucleophile residue capable of reacting with nitric oxide to form theN₂O₂ ⁻ functionality. Reaction of the biopolymer which contains asuitable nucleophilic residue, or of the suitably derivatizedbiopolymer, with nitric oxide thus provides a biopolymer-bound nitricoxide-releasing N₂O₂ ⁻ functional group.

To form the biopolymer-bound nitric oxide releasing N₂O₂ ⁻ functionalgroup, it is generally preferred to impart a net charge to the polymernear the site on the biopolymer where the N₂O₂ ⁻ functional group is tobe formed. By way of illustration, several general means are availablefor synthesizing a biopolymeric composition comprising a biopolymer towhich is attached a NONOate functional group. As one example, an ion ofstructure X—N₂O₂ ⁻ is reacted with an electrophilic agent (an[X′]⁺-donor) to generate a covalently bonded NONOate of formulaX—N(O)═NOX′; this protected complex is then attached to the desiredbiopolymer via the nucleophile residue, X, or the electrophile residue,X′. Alternatively, a nucleophile residue that is already part of (orthat can be attached to) the biopolymer can be reacted with NO underbasic conditions to give a nitric oxide complex containing a N₂O₂ ⁻functional group. As a specific example, a simple amino acid bearing asecondary amino group can be reacted with nitric oxide to generate acompound in accordance with the present invention. Similarly, theNONOate functionality can be attached to a basic nitrogen in a peptide.Alternative means can be used to attach NONOate-containing molecules tothiol or activated carboxylic acid groups in a peptide, polypeptide orprotein in accordance with the present invention.

Further, by way of illustration, the N₂O₂ ⁻ functional group may beattached to a peptide such as arg-gly-asp (RGD), to prepare the moleculearg-gly-asp-[N(O)NO]⁻. Preferably, the RGD tripeptide would be attachedto the NONOate through a linking group such as additional peptide units.Other receptor/ligand recognition sequences may be used analogously.

The biopolymer-bound nitric oxide-releasing compositions of the presentinvention will find utility in a wide variety of applications and in awide variety of forms depending on the biological disorder to betreated. For example, the biopolymer-bound NONOate may itself bestructurally sufficient to serve as implants, patches, stents,liposomes, microparticles, microspheres, beads, powders, liquids, gels,monolithic resins, disks, or the like, or the biopolymer-bound NONOatecan be attached to a non-biopolymer, or the like, suitable for suchpurpose. The term non-biopolymer is used herein to mean any polymer thatis not a biopolymer. Further, by way of illustration, thebiopolymer-bound NONOate composition can be incorporated into otherpolymer matrices, substrates or the like, or it may bemicroencapsulated, or the like.

The biopolymer-bound nitric oxide/nucleophile compositions of thepresent invention have a wide range of biological utility. In view ofthe growing awareness that nitric oxide is an especially versatile andimportant bioeffector species, having been implicated mechanistically insuch critical bodily functions as vasorelaxation, neurotransmission andthe immunological response (Moncada et al., Pharmacol. Rev., 43, 109-142(1991), the compositions of the present invention find utility inapplications where nitric oxide release is needed. For example, thebiopolymer-bound NONOates may be used to reduce the risk of restenosisafter angioplasty.

The following are further illustrative of, and not in any way inlimitation of, the broad uses and applications of the biopolymer-boundcompositions of this invention. Thus, for example, in view of dramaticbut short-lived pulmonary vaso- and bronchodilatory properties exhibitedby nitric oxide (Roberts et al., Circulation (Suppl. II), 84, A1279(1991)), administration of biopolymer-bound nitric oxide/nucleophileadduct compositions into the lungs in aerosolized form may be used intreating a variety of pulmonary disorders. Since natural, endogenousnitric oxide has been identified as an effector of penile erection(Blakeslee, New York Times, Jan. 9, 1992, page A1), the biopolymer-boundnitric oxide/nucleophile adduct compositions of the present inventionmay be incorporated into suitable penile implants, preparations fortransurethral injection, dermal patches or condoms for treatment ofimpotence in men. The ability of certain monomeric nitricoxide/nucleophile adducts to inhibit platelet aggregation coupled withtheir demonstrated cytostatic activity allows for an invaluabletwo-pronged approach to prevention of restenosis following angioplasty;stents fabricated with polymer-bound nitric oxide-releasing N₂O₂ ⁻functional group compositions may be used both to inhibit cell divisionin areas with damaged endothelium and to prevent adhesion of plateletsat these locations as well, minimizing the risk of recurring blockage.With an inverse relationship between generation of nitric oxide by tumorcells and their metastatic potential having been proposed (Radomski etal., Cancer Res., 51, 6073-6078 (1991), polymer-bound nitricoxide/nucleophile compositions can be used to reduce the risk ofmetastasis in cancer patients. Similarly, it is contemplated that thebiopolymer-bound nitric oxide-releasing compositions of the presentinvention can be used to coat prostheses and medical implants, such asbreast implants, prior to surgical connection to the body as a means ofreducing the risk of solid state carcinogenesis associated therewith.Tumor-specific antibodies containing the N₂O₂ ⁻ functional group can beused to sensitize cancer cells to radiotherapy. The N₂O₂ ⁻ functionalgroup can be attached to hormones that concentrate in the uterus, wherethe release of NO can halt premature labor. With nitric oxide beingadditionally implicated in gastric motility, neurotransmission,nociception, and other natural roles, the compositions of this inventioncan be used for those applications as well.

In another aspect of the invention, there is provided a polymer-boundnitric oxide-releasing composition which comprises the novelbiopolymer-bound nitric oxide-releasing compositions of the presentinvention and a-non-biopolymer as disclosed in copending applicationSer. No. 07/935,565. In accordance with this aspect of the invention,the biopolymer-bound NONOates described herein are incorporated into orbonded to a non-biopolymer. For this use, any of a wide variety ofpolymers can be used. It is only necessary that the polymer selected isbiologically acceptable. Illustrative of polymers suitable for use inthe present invention are polyolefins, such as polystyrene,polypropylene, polyethylene, polytetrafluorethylene, polyvinylidenedifluoride, and polyvinylchloride, polyethylenimine or derivativesthereof, polyethers such as polyethyleneglycol and polysaccharides,polyesters such as poly(lactide/glycolide), polyamides such as nylon,polyurethanes, colestipol and derivatives thereof. The biopolymericnitric oxide-releasing compositions described above may be bound to anon-biopolymer support in a number of different ways. For example, thebiopolymer-bound NONOates may be bound to the non-biopolymer bycoprecipitation of the biopolymer with the non-biopolymer.Coprecipitation involves, for example, solubilizing both thenon-biopolymer and the biopolymer-bound NONOate and evaporating thesolvent. Alternatively, the biopolymer-bound NONOates can be chemicallybonded to the non-biopolymer.

The physical and structural characteristics of the non-biopolymerssuitable for use in the present invention are not narrowly critical, butrather will depend on the end use application. It will be appreciated bythose skilled in the art that where the resulting polymeric compositionis intended for topical, dermal, percutaneous, or similar use, it neednot be biodegradable. For some uses, such as ingestion or the like, itmay be desirable that the non-biopolymer slowly dissolve in aphysiological environment or that it is biodegradable. The resultingpolymeric forms can be bioerodible, durable or instantly soluble inphysiological fluids.

One skilled in the art will appreciate that suitable methods ofadministering the biopolymer-bound nitric oxide-releasing N₂O₂ ⁻functional group compositions of the present invention to an animal areavailable, and, although more than one route can be used to administer aparticular composition, a particular route can provide a more immediateand more effective reaction than another route. Pharmaceuticallyacceptable carriers are also well-known to those who are skilled in theart. The choice of carrier will be determined in part by the particularcomposition, as well as by the particular method used to administer thecomposition. Accordingly, there is a wide variety of suitableformulations of the pharmaceutical composition of the present invention.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the biopolymer-boundcomposition dissolved in diluents, such as water or saline, (b)capsules, sachets or tablets, each containing a predetermined-amount ofthe active ingredient, as solids or granules, (c) suspensions in anappropriate liquid, and (d) suitable emulsions. Tablet forms can includeone or more of lactose, mannitol, corn starch, potato starch,microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide,croscarmellose sodium, talc, magnesium stearate, stearic acid, and otherexcipients, colorants, diluents, buffering agents, moistening agents,preservatives, flavoring agents, and pharmacologically compatiblecarriers. Lozenge forms can comprise the active ingredient in a flavor,usually sucrose and acacia or tragacanth, as well as pastillescomprising the active ingredient in an inert base, such as gelatin andglycerin or sucrose and acacia emulsions, gels, and the like containing,in addition to the active ingredient, such carriers as are known in theart.

The biopolymer-bound nitric oxide-releasing compositions of the presentinvention, alone or in combination with other suitable components, canbe made into aerosol formulations to be administered via inhalation.These aerosol formulations can be placed into pressurized acceptablepropellants, such as dichlorodifluoromethane, propane, nitrogen, and thelike.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

The dose administered to an animal, particularly a human, in the contextof the present invention should be sufficient to effect a therapeuticresponse in the animal over a reasonable time frame. The dose will bedetermined by the strength of the particular compositions employed andthe condition of the animal, as well as the body weight of the animal tobe treated. The size of the dose also will be determined by theexistence, nature, and extent of any adverse side-effects that mightaccompany the administration of a particular composition.

The following examples further illustrate the present invention, but donot limit the scope thereof.

In the Examples, chemiluminescence analysis for total recoverable nitricoxide from polymers containing the nitric oxide-releasing N₂O₂ ⁻functional group by acid treatment was carried out as follows:

The analysis of NO adducts, i.e., polymers containing the nitricoxide-releasing N₂O₂ ⁻ functional group, was done on a nitric oxideanalyzer and was patterned after the procedure of Maragos et al., J.Med. Chem., 34, 3242-3247 (1991). A reactor vessel fitted with a septumwas charged with a small aliquot of the polymer to be studied and thesystem was purged with helium for several minutes to remove traces ofoxygen. Two milliliters of 10 mM sulfuric acid was added by injectionthrough the septum to begin reaction. Gaseous effluent was sweptcontinuously via a fritted glass bubbler positioned at the bottom of thereactor vessel (i.e., immersed in the acid solution) into achemiluminescence detector (Thermal Energy Analyzer Model 502LC,Thermedics, Inc., Woburn, Mass.). The area of the resultingchemiluminescence signal versus time curve was electronically computedand compared with that of a known quantity of nitric oxide gas standardto determine the amount of nitric oxide produced by acid treatment ofthe polymer aliquot.

This procedure was used to estimate the total amount of nitric oxiderecoverable from the polymer. To estimate the rate of nitric oxidegeneration under physiological conditions, the inventive polymers weresubjected to a procedure identical to that described above except that 2ml of 10 mM phosphate buffer, pH 7.4, at 37° C. was injected into thereactor vessel in place of the sulfuric acid solution to start thereaction.

EXAMPLES

The preparation and characterization of biopolymers containing thenitric oxide-releasing N₂O₂ ⁻ functional group are illustrated in thefollowing examples:

Example I

This example illustrates the preparation of1-(2S-carboxypyrrolidin-1-yl)-1-oxo-2-hydroxydiazene, disodium salt, asshown schematically as follows:

A solution of 10 g (0.087 mol) of L-proline in 39 ml (0.18 mol) of 25%sodium methoxide in methanol and 20 ml of methanol was degassed andexposed to 40 psi of NO for 20 h. The pressure was released and thesolid residue was collected by filtration, washed with ether, and driedunder vacuum to give 17 g of a white solid: UV (0.01 M NaOH) λ_(max) (ε)250 nm (ε=4.9 mM⁻¹ cm⁻¹); NMR (D₂O) δ 1.71 (m, 1 H), 1.91 (m, 2 H), 2.27(,m, 1 H), 3.27-3.43 (m, 2 H), 4.04 (m, 1 H). A methanol peak was alsopresent, but the solid was free of both proline and N-nitrosoproline.

Example II

This example illustrates the preparation of1-hydroxy-2-oxo-3-carboxymethyl-3-methyl-1-triazene, disodium salt, asshown schematically as follows:

To a solution of 8 g (0.2 mol) of sodium hydroxide in 100 ml of methanoland 20 ml of water was added 8.9 g (0.1 mol) of sarcosine. The solutionwas charged with 40 psi of NO and stirred at 250° C. for 48 h. Thepressure was released, and the solution was evaporated in vacuo to givea white solid: UV λ_(max) 250 nm. The distillate had a strong amineodor, which was determined to be methylamine on derivatization withbenzoyl chloride.

The solid residue was dried under high vacuum, then analyzed by NMR inD₂O. Five products were detected by NMR: methylamine, δ 2.28, 36%;1-dimethylamino-1-oxo-2-hydroxydiazene, sodium salt, δ 2.79, 15%;N-nitrosodimethylamine, δ 3.11 and 3.91, 8%; N-nitrososarcosine, sodiumsalt, δ 3.15 (s, E methyl), 3.84 (s, Z methyl), 4.21 (s, Z methylene),4.80 (s, E methylene), 10%. The title compound was present as 32% of themixture: δ 3.11 (s, 3 H) and 3.60 (s, 2 H).

Example III

This example illustrates the preparation of1-hydroxy-2-oxo-3-carboxymethyl-3-methyl-1triazene N-methylamide, sodiumsalt, as shown schematically as follows:

A solution of 150 ml (1.9 mol) of 40% aqueous methylamine was cooled to0° C. To the solution was added 40 ml of 10 M sodium hydroxide followedby the careful addition over a 2-h period at 0° C. of α-chloroacetylchloride (27 g, 0.24 mol).

Stirring was continued at room temperature overnight. The resultingsolution was saturated with sodium chloride and extracted withdichloromethane, dried over sodium sulfate, and filtered through a layerof magnesium sulfate. Most of the solvent was removed on a rotaryevaporator and the residue was distilled at 1 atm then under moderatevacuum. The product distilled at 90-2° C. at 125 mm Hg to yield 15 g(61%) of sarcosine N-methylamide: IR (film) 3318, 2952, 2889, 1659,1553, 1462, 1413, 1166 cm⁻¹; NMR (CDCl₃) δ 2.42 (s, 3 H), 2.86 s 1.5 H),2.83 (s, 1.5 H), 3.23 (s, 2 H).

A solution of 1.7 g (0.0167 mol) of sarcosine N-methylamide in 3.5 ml(0.016 mol) of 25% sodium methoxide in methanol was placed in a pressurebottle, flushed with nitrogen and charged with 40 psi of nitric oxide.The solution was kept at 25° C. for 48 h, giving a thick paste. Thepressure was released. The residue was washed with ether and dried undervacuum to give 1.4 g of a solid: UV λ_(max) (ε) 250 nm (2.4 mM⁻¹ cm⁻¹).

Example IV

This example illustrates the preparation of the bis(nitric oxide) adductof L-prolyl-L-leucylglycinamide, as shown schematically as follows:

To a slurry of 120 mg (0.423 mmol) of L-prolyl-L-leucylglycinamide(Sigma) in 4 ml of acetonitrile was added 100 μl of 25% sodium methoxidein methanol. The resulting gel was treated with a few drops of methanoluntil a homogeneous solution was obtained. The solution was transferredinto a micro-Parr bottle and bubbled with nitrogen for 5 min, followedby exposure to 40 psi of NO for 72 h. The reaction mixture was driedunder vacuum to give 187 mg of a solid: λ_(max) (ε) 250 nm (6.2 mM⁻¹cm⁻¹) in pH 7.4 buffer. It released 0.86 moles of NO (per mole oftripeptide decomposed at this pH) with a half-life of 7 min at 37° C.

Oligopeptides and proteins of increasing chain length can be similarlyderivatized with NO.

Example V

This example demonstrates the attachment of a nucleophilic center to aprotein that does not contain a nucleophilic center that will readilyreact with NO, shown schematically as follows:

A solution of 4.78 g (0.025 mol) of N-acetyl-L-methionine in CH₂Cl₂:acetonitrile (120 ml) was cooled to 0° C. To this solution was added5.36 g (0.025 mol) of dicyclohexylcarbodiimide (DCC) followed by therapid addition of 3.90 g (0.021 mol) of N-t-butoxycarbonylpiperazine in6 ml of dichloromethane. The progress of the reaction was followed onsilica gel TLC plates developed with 4:1 acetonitrile: tetrahydrofuranand visualized with either iodine or ninhydrin spray. The reaction wascomplete within 2 h. A few drops of glacial acetic acid were added tothe reaction mixture and the solvent was removed on a rotary evaporator.The residue was taken up in ether and filtered. The clear filtrate waswashed with dilute acid followed by dilute base. The organic layer wasseparated, dried over anhydrous sodium sulfate, filtered, and evaporatedto give 8.2 g of 1-(t-butoxycarbonyl)-4-(N-acetyl-L-methionyl)piperazine, a colorless oil which required nofurther purification: IR (film) 3304, 3058, 2973, 2931, 2868, 1701,1645, 1539, 1420, 1237, 1173 cm⁻¹; NMR (CDCl₃) δ 1.47 (s, 9 H), 1.80 (m,2 H), 2.02 (s, 3 H), 2.10 (s, 3 H), 2.46 (m, 2 H), 3.53 (m, 8 H), 5.10(M, 1 H), 6.35 (b, 0.5 H), 6.43 (b, 0.5 H).

To a solution of 8.6 g (0.024 mol) of 1-(t-butoxycarbonyl)-4-(N-acetyl-L-methionyl)piperazine in 60 ml of dichloromethane wasadded 10 ml of trifluoroacetic acid and the mixture was stirred at roomtemperature overnight. The solution was extracted with water and theresulting aqueous solution was made basic with sodium hydroxide. Theproduct was extracted with dichloromethane, dried over sodium sulfate,and filtered. Evaporation of the solvent gave 2.1 g of1-(N-acetyl-L-methionyl)piperazine, as an oil: IR (film) 3304, 3051,2917, 2861, 1645, 1546, 1448, 1377 cm⁻¹; NMR (CDCl₃) δ 1.95 (m, 2 H),2.02 (s, 3 H), 2.10 (s, 3 H), 2.54 (m, 2 H), 2.98 (m, 4 H), 3.74 (m, 4H), 5.10 (m, 1 H), 6.40 (b, 0.5 H), 6.48 (b, 0.5 H).

To a solution of 510 mg (1.97 mmol) of1-(N-acetyl-L-methionyl)piperazine in 1 ml of methanol was added 428 μl(1.97 mmol) of 25% sodium methoxide in methanol. The system was degassedand charged with 40 psi of nitric oxide. After exposure of the solutionto NO for 120 h, the pressure was released and the solid product wascollected by filtration, washed with ether, and dried to give 27 mg of1-[4-(N-acetyl-L-methionyl)piperazin-l-yl]-1-oxo-2-hydroxydiazene,sodium salt, as a white solid: UV λ_(max) (ε) 252 nm (12.0 mM⁻¹ cm⁻¹).The product decomposed with a half-life of 6.9 min at pH 7 and 25° C. toproduce 1.72 moles of NO per mole of test agent.

Example VI

This example demonstrates the attachment of a preformed NONOatecontaining a nucleophilic nitrogen atom to the C-terminus of a peptide,polypeptide or protein as shown schematically as follows:

A solution of 20 g (0.126 mol) of ethyl 1-piperazinecarboxylate in 60 mlof methanol was placed in a Parr bottle. The solution was treated with27.4 ml (0.126 mol) of 25% sodium methoxide in methanol. The system wasevacuated, charged with 40 psi of nitric oxide and kept at 25° C. for 48h. The white crystalline product was collected by filtration and washedwith cold methanol as well as with copious amounts of ether. The productwas dried under vacuum to give a 14.5 g (48%) yield of1-(4-carbethoxypiperazin-1-yl)-1-oxo-2-hydroxydiazene, sodium salt: mp184-5° C.; UV (0.01 M NaOH) λ_(max) (ε) 252 nm (10.4 mM⁻¹ cm⁻¹); NMR(D₂O) δ 1.25 (t, 3 H), 3.11 (m, 2 H), 3.68 (m, 2 H), 4.15 (q, 2 H). AnalCalcd. for C₆H₁₃N₄O₄Na: C, 35.00%; H, 5.42%; N, 23.33%; Na, 9.58%.Found: C, 34.87%; H, 5.53%; N, 23.26%; Na, 9.69%. The half-life of thiscompound at pH 7 and 25° C. was 5 min. This measurement was based on theloss of the 252-nm chromophore in the ultraviolet spectrum.

A solution of 1.3 g (5.4 mmol) of1-(4-carbethoxypiperazin-1-oxo-2-hydroxydiazene, sodium salt, in 10 mlof 0.01 M aqueous sodium hydroxide was cooled in an ice bath. A solutionof 2 ml of dimethyl sulfate in 10 ml of methanol was added dropwise. Theresulting solution was stirred at 0° C. for 1 h, then allowed to warmgradually to room temperature. After 24 h the solution was concentratedon a rotary evaporator. The residue was extracted with dichloromethane,dried over sodium sulfate, and filtered through a layer of magnesiumsulfate. The solvent was evaporated under reduced pressure and theresidue was chromatographed on silica gel. Elution with 2:1dichloromethane:ethyl acetate provided 683 mg (55%) of1-(4-carbethoxypiperazin-1-y)-1-oxo-2-methoxydiazene as an oil, whichcrystallized on standing: mp 46° C.; UV λ_(max) (ε) 240 nm (8.4 mM⁻¹cm⁻¹); IR (film) 2988, 2945, 2875, 1707, 1504, 1068 cm⁻¹; NMR δ 3.38 (m,4 H), 3.67 (m, 4 H), 4.03 (s, 3 H), 4.16 (q, 2 H); MS m/z (relativeintensity, %), 232 (M⁺, 3), 217 (16), 187 (10), 157 (100), 142 (5), 98(4), 85 (27), 70 (26), 56 (94), 54 (19); exact mass calcd for C₈H₁₆N₄O₄(M⁺) 232.1171, found 232.1172. Anal Calcd for C₈H₁₆N₄O₄: C, 41.38%; H,6.90%; N, 24.14%. Found C, 41.23%; H, 6.82%; N, 24.05%.

A mixture of 1.8 g (0.0078 mol) of1-(4-carbethoxypiperazin-1-yl)-1-oxo-2-methoxydiazene and 20 ml of 5 Maqueous sodium hydroxide was heated at reflux. After 45 min no startingmaterial remained in the mixture, as assessed from qualitative thinlayer chromatography. The solution was allowed to cool to roomtemperature and evaporated to a viscous residue, which was extractedwith ethyl acetate, dried over sodium sulfate, filtered, and evaporated.The product was chromatographed on silica gel and eluted with 1:1dichloromethane:acetone giving 820 mg (66%) of1-(piperazin-1-yl)-1-oxo-2-methoxydiazene as a pale yellow oil: UVλ_(max) (ε) 234 nm (7.0 mM⁻¹ cm⁻¹); NMR δ 3.03 (m, 4 H), 3.38 (m, 4 H),4.06 (s, 3 H); IR (film) 3318, 2945, 2854, 1447, 1364, 1286, 1230, 1046,1004 cm⁻¹; MS m/z (relative intensity, %) 160 (M⁺, 2), 145 (7), 143(10), 115 (9), 85 (56), 58 (7), 56 (100); exact mass calcd for C₅H₁₂N₄O₂(M⁺) 160.0960, found 160.0966.

To a solution of 164 mg (0.856 mmol) of N-acetyl-L-methionine in 10 mlof 1:1 dichloromethane:acetonitrile was added 206 mg (1 mmol) ofdicyclohexylcarbodiimide (DCC) followed by the rapid introduction of 137mg (0.856. mmol) of 1-(piperazin-1-yl)-1-oxo-2-methoxydiazene in 3 ml ofdichloromethane. The reaction mixture was stirred at 25° C. for 4 h. Afew drops of glacial acetic acid were added to decompose excess DCC. Themixture was filtered and evaporated. The residue was extracted withethyl acetate, which in turn was washed with dilute hydrochloric acid,followed by dilute aqueous sodium hydroxide. The organic layer was driedover sodium sulfate, filtered through, a layer of magnesium sulfate, andevaporated in vacuo. Purification of1-(4-[N-acetyl]-L-methionylpiperazin-1-yl)-1-oxo-2-methoxydiazene wasaccomplished on silica gel using 4:1 acetonitrile:tetrahydrofuran as theeluant: UV λ_(max) (ε) 230 nm (8.7 mM⁻¹ cm⁻¹); NMR δ 2.02 (s, 3 H), 2.07(m, 2 H), 2.11 (s, 3 H), 3.46 (m, 4 H), 3.83 (m, 4 H), 4.03 (s, 3 H),5.15 (m, 1 H), 6.28 (b, 0.5 H), 6.35 (b, 0.5 H); IR 3297, 2931, 2847,1645, 1546, 1497, 1441, 1223 cm⁻¹; MS m/z (relative intensity, %), 333(M⁺, 4), 318 (2), 304 (3), 303 (16), 288 (12), 260 (11), 259 (100), 258(9), 214 (78), 184 (37), 183 (10), 174 (5), 146 (26), 142 (56), 141 (5),104 (63), 61 (60); exact mass calcd for C₁₂H₂₃N₅O₄S (M⁺) 333.1470, found333.1471.

All publications, patents, and patent applications cited-herein arehereby incorporated by reference to the same extent as if eachindividual document were individually and specifically indicated to beincorporated by reference and were set forth in its entirety herein.

While this invention has been described with emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat the preferred embodiments may be varied. It is intended that theinvention may be practiced otherwise than as specifically describedherein. Accordingly, this invention includes all modificationsencompassed within the spirit and scope of the appended claims.

1. An implant or intravascular stent comprising a polymeric compositioncapable of releasing nitric oxide, said composition comprising a polymerand at least one nitric oxide-releasing N₂O₂ ⁻ functional group bound tosaid polymer.
 2. An intravascular stent comprising a nitricoxide-releasing agent, wherein said agent is selected from the groupconsisting of X

N(O)NO] and [N(O)NO

X, wherein X is an organic or inorganic moiety, and wherein said agentis present in an amount effective to reduce restenosis in a mammal. 3.An intravascular stent comprising a polymeric composition capable ofreleasing nitric oxide, said composition comprising at least one nitricoxide-releasing N₂O₂ ⁻ functional group bound to said polymer, whereinsaid composition is present in an amount effective to reduce restenosisin a mammal.
 4. An intravascular stent comprising an amount of acytostatic therapeutic agent, wherein the agent is a nitricoxide-releasing agent selected from the group consisting of X

N(O)NO] and [N(O)NO

X, wherein X is an organic or inorganic moiety agent, which agent doesnot exhibit substantial cytotoxicity, which agent is effective to reducerestenosis following placement of the stent, wherein the therapeuticagent is not heparin or a radioisotope.
 5. An intravascular stentcomprising an amount of a nitric oxide-releasing agent which does notexhibit substantial cytotoxicity, which agent is effective to reducerestenosis following placement of the stent, wherein the agent is notheparin or a radioisotope.